Penicillin acylases (E.C. 3.5.1.11, penicillin amidohydrolase) are produced by bacteria, actinomycetes, fungi and yeast. These important industrial enzymes are on the basis of their substrate specificity divided into three groups: penicillin G acylases (PGA), penicillin V acylases (PVA) and ester hydrolases of α-amino acids (AEH, formerly named ampicillin acylases). The enzymes of the PGA group have broad substrate specificity and catalyse the hydrolysis of the amidic bond of penicillins and cephalosporins. Among the bacterial producers of the PGA belong the species of the genera: Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Corynebacterium, Escherichia, Erwinia, Flavobacterium, Kluyvera, Micrococcus, Nocardia, Proteus, Providencia, Pseudomonas, Sarcina, Xanthomonas, Xylella (Process Biochem. 24:146-154, 1989, Process Biochem. 27:131-143, 1992, Biotechnol. Adv. 18:289-301, 2000).
Apart from the production strains expressing PGA, obtained by the mutagenesis, recombinant microorganisms containing recombinant plasmids with the structural gene encoding PGA were prepared from Escherichia coli (CS 244 343 and CS 246 957), Alcaligenes sp. or fecalis (Current Microbiology 39:2444-2448, 1999; EP 638649, Appl. Environ. Microbiol. 63: 3412-3418, 1997), Arthrobacter viscosus (Appl. Environ. Microbiol. 54: 2603-2607, 1988 a 55: 1351-1356, 1989; Gene 143: 79-83, 1994), Bacillus megaterium (J. Bacteriol. 93: 302-306, 1967, Appl. Microbiol. Biotechnol. 25: 372-378, 1987; U.S. Pat. No. 3,145,395; Process Biochemistry 29: 263-270, 1994), Kluyvera citrophila (Agric. Biol. Chem. 39: 1225-1232, 1975; Gene 49: 69-80, Biotechnol. Letters 14: 285-290, 1992), Providencia rettgeri (Acta Biochim. Polonica 28: 275-284, 1981; J. Bacteriol. 168: 431-433, 1986; DNA sequences 3: 195-200, 1992).
Most prokaryotic producers of the PGA are gram-negative bacteria and the enzyme is located in the periplasma of the cell. In the Bacillus megaterium culture, the enzyme is secreted from the cell to the medium.
The penicillin G acylases are industrially used mainly for the hydrolysis of the phenylacetyl derivatives of cephalosporins and penicillin G for the purpose of the preparation of the intermediates 6-APA and 7-ADCA. At present, these enzymes are used also in synthetic reactions, in the acylations of the above-mentioned intermediates, leading to the preparation of semi-synthetic antibiotics (e.g., U.S. Pat. No. 5,753,458 and U.S. Pat. No. 5,801,011; WO 98/04732; WO 97/04086; Enzyme Microb. Technol. 25: 336-343, 1999; Synthesis of β-lactam antibiotics: Chemistry, Biocatalysis and Process Integration, Ed.: A. Bruggink, Kluwer Academic Publishers, Dordrecht/Boston/London, 2001).
For a repeated, long-term use of the PGA in the catalysis of the enzyme reaction, the enzyme is stabilized by immobilization or encapsulation, forming an enzyme catalyst. In this form, the enzyme shows a higher pH stability, temperature stability and a longer half-life period under the reaction conditions, under which it catalyzes the course of the reaction.